目的 基于药物亲和反应的靶点稳定性分析技术（DARTS）及液相色谱串联质谱法（LC-MS/MS）研究黄芪甲苷（ASI）在HK-2（肾小管上皮细胞）上的作用靶标，并进行验证。方法 采用40 mmol·L^-1葡萄糖干预HK-2细胞24 h，制备糖尿病肾病（DKD）细胞模型；将HK-2细胞随机分为：对照组、甘露醇（等渗对照）组、模型组和ASI低、中、高浓度（1.6、3.2、6.4 μmol·L^-1）组，造模后加药干预24 h； CCK-8法检测细胞存活率，流式细胞仪检测细胞凋亡，ELISA法检测细胞上清液中肿瘤坏死因子（TNF）-α、血管内皮生长因子（VEGF）、肾损伤分子-1（KIM-1）水平。运用DARTS技术联合定量蛋白组学分析筛选出肾小管上皮细胞中可能与ASI发生结合的蛋白。基于差异蛋白筛选结果，与GeneCard、DisGeNET和OMIM数据库中筛选的“糖尿病肾病”“细胞凋亡”靶标取交集，选取与DKD相关的蛋白，采用分子对接、DARTS联合Western blotting实验及生物膜干涉技术对其进行验证。结果 与模型组比，ASI能提高高糖诱导下的HK-2细胞活力，并抑制细胞凋亡及TNF-α、VEGF、KIM-1的分泌（P＜0.05、0.01）。DARTS联合定量蛋白组学分析鉴定出32个差异蛋白，其中线粒体凋亡诱导因子1（AIFM1）、2,4-双烯酰辅酶A还原酶1（DECR1）与“糖尿病肾病”“细胞凋亡”相关；分子对接显示，ASI与AIFM1的结合活性更强； DARTS-Western blotting实验显示，ASI能提高AIFM1的酶解稳定性；生物膜干涉实验显示，ASI与AIFM1具有良好的亲和性。结论 ASI可能通过靶向作用于HK-2细胞上的AIFM1发挥细胞保护作用。

Objective To investigate the target proteins of astragaloside IV (ASI) in HK-2 cells (renal tubular epithelial cells) using drug affinity responsive target stability (DARTS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), and to validate these targets. Methods HK-2 cells were treated with 40 mmol·L^-1 glucose for 24 h to establish a diabetic kidney disease (DKD) cell model. Cells were randomly divided into control, mannitol (isotonic control), model, and ASI low-, medium-, and high-dose groups (1.6, 3.2, and 6.4 μmol·L^-1), respectively. After modeling, drugs were added and incubated for 24 h. Cell viability was assessed by CCK-8 assay, apoptosis was analyzed by flow cytometry, and levels of tumor necrosis factor-alpha (TNF-α), vascular endothelial growth factor (VEGF), and kidney injury molecule-1 (KIM-1) in cell supernatants were measured by ELISA. DARTS combined with quantitative proteomics was used to identify potential proteins interacting with ASI in renal tubular epithelial cells. Based on differentially expressed proteins, intersection analysis was performed with targets related to “diabetic kidney disease” and “apoptosis” from GeneCard, DisGeNET, and OMIM databases to select DKD-related proteins. These candidates were further validated through molecular docking, DARTS-Western blotting, and bioluminescence resonance energy transfer (BRET) assays. Results Compared with the model group, ASI significantly improved cell viability, inhibited apoptosis, and reduced secretion of TNF-α, VEGF, and KIM- 1 under high-glucose conditions (P < 0.05, 0.01). DARTS combined with quantitative proteomics identified 32 differentially expressed proteins, among which mitochondrial apoptotic inducer 1 (AIFM1) and 2,4-dienoyl-CoA reductase 1 (DECR1) were associated with “diabetic kidney disease” and “apoptosis”. Molecular docking showed stronger binding affinity between ASI and AIFM1. DARTSWestern blotting revealed that ASI enhanced the enzymatic stability of AIFM1. BRET experiments confirmed strong binding affinity between ASI and AIFM1. Conclusion ASI may exert its protective effects on HK-2 cells by targeting AIFM1.

R965

贵州省卫生健康委科学技术基金项目（gzwkj2024-514）；贵州省基础研究计划（自然科学）面上项目（黔科合基础MS[2025]172）；贵州省中医药管理局中医药、民族医药科学技术研究课题（QZYY-2025-012，QZYY-2026-096）；贵州省科技计划项目（黔科合基础MS［2026］647）；贵州中医药大学青年扬帆计划（贵中医科合-QNYFZK［2025］16号）